Effect of light exposure on circadian rhythm in theront excystment in Cryptocaryon irritans.

We examined the effects of light exposure on the theront excystment circadian rhythm in Cryptocaryon irritans using a newly invented apparatus, which enabled us to examine the excystment rhythms of theronts from tomonts with low labor. Using the apparatus, we examined the timings of theront excystment from tomonts exposed directly to light and from tomonts exposed to light-exposed seawater by counting the number of excysted theronts. We found that the theront excystment time changed only when tomonts were directly exposed to light, indicating that light reception is essential for circadian rhythm entrainment. When tomonts were exposed to light only once for 12 h, either on Day 1, Day 2, or Day 3 after leaving host and being encysted, the circadian rhythm was entrained according to the photoperiod given on tomonts. Tomonts exposed to a low light (1 lx) with 12L:12D photoperiod daily showed a circadian rhythm similar to that in tomonts exposed to an intense light (500 lx) under the same photoperiod. When tomonts were incubated at 22 °C, 25 °C, or 28 °C under the same photoperiod, almost the same circadian rhythm was developed, suggesting temperature has little effect on the circadian rhythm entrainment between the range, even though the date of excystment was delayed in lower temperatures. These results suggest the circadian rhythm of theront excystment can be entrained in tomonts on the seabed of inner bays where net-cage aquaculture is conducted, and be involved in the outbreaks of cryptocaryoniasis there.

Effect of Biokos, a natural lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on infection of Cryptocaryon irritans.

Cryptocaryoniasis (marine white spot disease), caused by Cryptocaryon irritans, is a major threat to marine fish cultures in tropical and subtropical waters, and a serious nuisance to hobbyists with saltwater fish tanks. With only classical treatment schedules such as copper salts or hyposaline baths being available, control of the disease remains a challenge. In this study, we investigated the effect of Biokos, a viscosin-like lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on the external life stages of C. irritans, including theronts, protomonts and tomonts. The present study demonstrated that the compound has an antiparasitic effect on all tested external stages of the parasite. In particular, when Biokos was used at 48 mg/L, it was able to kill almost all theronts and protomonts within 1 h in in vitro experiments, and using the same concentration in an in vivo challenge experiment, the parasitic load was reduced by more than 95% compared to the control group with no Biokos. Additionally, cultured fish cells were able to proliferate, and fish showed no adverse signs at Biokos concentrations that were effective in killing the parasite. Thus, Biokos may be a promising way for preventing or reducing the burden of this parasitic disease in the future.

Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Target Protein Expression on Tetrahymena thermophila Cell Surface Using the Signal Peptide and GPI Anchor Sequences of the Immobilization Antigen of Cryptocaryon irritans.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites".

Effects of temperature on Anisakis simplex sensu stricto L3 larvae experimentally challenged in rainbow trout (Oncorhynchus mykiss) and Mozambique tilapia (Oreochromis mossambicus).

Human anisakiasis is a foodborne disease that has been increasingly reported worldwide and is caused by the consumption of raw or undercooked seafood infected with zoonotic nematodes of the genus Anisakis Dujardin, 1845. One of the most frequently reported species, both in fish paratenic hosts as well as in human patients, is Anisakis simplex sensu stricto (s.s.), which is distributed within some of the globe's main fishing grounds in the North Pacific and Atlantic oceans. In order to clarify the influence of temperature on this parasite in vivo, third-stage larvae of A. simplex s.s. were surgically challenged in the body cavities of rainbow trouts (Oncorhynchus mykiss) and Mozambique tilapias (Oreochromis mossambicus). Larval survival and tissue migration were then analyzed after 6 and 12 weeks. The results showed that survival rates of larvae were lower at 27 and 33 °C than at 3, 9, 15 and 21 °C. Also, migration to the body muscle was observed to be highest at 9 °C. These results suggest that third-stage larvae of A. simplex s.s. are more adapted to lower temperatures in experimentally challenged fish, which may justify its distribution in cold northern waters.

Effects of temperature on eggs and larvae of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) and its possible role on their geographic distributions.

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3-25 °C and those of A. pegreffii hatched at 3-27 °C. Days before hatching varied between 2 days at 25 °C and 35-36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.

Establishment and characteristics of Perkinsus beihaiensis in vitro isolates from Mediterranean mussels Mytilus galloprovincialis.

Two monoclonal in vitro isolates of Perkinsus beihaiensis were established from tissues of infected Mediterranean mussels Mytilus galloprovincialis, using a lipid concentrate and yeast extract medium (LpcYM) to induce enlarged prezoospoangia from histozoic trophozoites, and Perkinsus broth medium (PBM) to support subsequent zoosporulation and schizogonic proliferation from those cells. Zoospores of the isolates developed into uninucleate signet-ring trophozoites followed by schizonts typical of Perkinsus spp., but an additional type of unknown botryoidal cell clusters appeared and increased in density. These botryoidal cell clusters had second-generation daughter schizonts within which further internal cells were produced. Although botryoidal cell clusters in primary cultures rarely proliferated in PBM, cells contained by botryoidal clusters enlarged as prezoosporangia in LpcYM, and a large number of proliferative stages (i.e. trophozoites and schizonts typical for Perkinsus spp.) were obtained from these prezoosporangia in PBM. At lower cell density, trophozoites transformed into the botryoidal cell clusters in PBM, and the number of cells increased little, while trophozoites rapidly increased by typical schizogony at higher density in PBM. Based on these observations, we determined an optimal cell density for continuous culture of P. beihaiensis isolates, confirmed their ability to proliferate and survive after cryopreservation, and describe an experimental protocol to culture this most recently described species in the genus Perkinsus.

Morphological and molecular characterization of Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae) from the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam.

A new myxozoan species, Ceratomyxa binhthuanensis n. sp. (Myxosporea: Ceratomyxidae), was found in the gall bladder of blacktip grouper Epinephelus fasciatus (Perciformes: Serranidae) in the East Sea of Vietnam. Myxospores were observed floating free in the gall bladder of 3 out of 20 fish examined (15%). Mature myxospores were elongate and slightly crescent-shaped and measured 12.2 ± 1.3 (10.8-16.0) µm in thickness and 5.8 ± 0.6 (4.8-6.9) µm in length, with two smooth equal shell valves. The two polar capsules were spherical and equal in size, measuring 2.6 ± 0.3 (2.3-2.9) µm in diameter. The posterior angle was slightly concave, 153.7° ± 5.6° (148.9°-166.0°). Molecular analysis of SSU rDNA sequence showed that Ceratomyxa binhthuanensis n. sp. differs from other Ceratomyxa spp. available in GenBank. Phylogenetic analysis indicated that C. binhthuanensis n. sp. was closely related to three species, Ceratomyxa nolani, Ceratomyxa yokoyamai, and Ceratomyxa cutmorei, which also infect fish hosts of the genus Epinephelus.

Supplementation with lipids enhances zoosporulation of Perkinsus species.

The protozoan parasite Perkinsus olseni is a major pathogen of marine mollusks. A large number of zoospores (the most infective stage) is required to experimentally examine the transmission and invasion processes of this parasite, but the conventional zoosporulation method in seawater is inefficient. A recent study found that P. olseni zoosporulation was highly enhanced in a nutrient-rich medium which was developed for trophozoite proliferation in Perkinsus. Here, we examined the effect of each medium component on P. olseni zoosporulation and found that lipids enhanced zoosporulation. Moreover, zoospores failed to develop into trophozoites in seawater supplemented with lipids, indicating that this zoosporulation method using seawater supplemented with lipids is ideal for preparing a large number of P. olseni zoospores for various biological assays. Lipid supplementation also significantly enhanced zoosporulation in P. honshuensis, P. chesapeaki, and P. marinus, although the enhancing effect of the lipid supplementation varied by species. Considering the limited availability of lipids in the sea water column, our findings suggest that Perkinsus zoosporulation likely occurs mainly in the vicinity of hosts (not in the sea water column), such as in the mantle cavity or on the body surface of mollusk hosts.

Francisellosis of Yesso scallops Mizuhopecten yessoensis in Japan is caused by a novel type of Francisella halioticida.

Francisella halioticida, the causative agent of francisellosis of the giant abalone Haliotis gigantea, has also been isolated from Yesso scallops Mizuhopecten yessoensis, which presented with orange/pinkish lesions in the adductor muscle and experienced high mortality. However, it is not clear whether the F. halioticida isolated from the giant abalone and Yesso scallops are phenotypically and genetically identical to each other. The present study revealed that isolates from the giant abalone and Yesso scallops were phenotypically different, with slower growth in modified eugon broth and a lack of prolyl aminopeptidase and phenylalanine aminopeptidase in Yesso scallop isolates. Additionally, we found that 3 of 8 housekeeping genes were different between them. Based on these phenotypic and genetic differences, we propose that F. halioticida isolated from Yesso scallops in Japan be designated as the 'J-scallop type' to distinguish it from strains from abalone ('abalone type'). Whole-genome sequencing analysis of a strain belonging to the J-scallop type showed that the overall similarity between the J-scallop and abalone type strains was estimated to be 99.84%. In accordance with a lack of prolyl aminopeptidase activity, in general, all of the J-scallop type strains examined have a 1 bp deletion in the responsible gene encoding prolyl aminopeptidase. This deletion was confirmed in all F. halioticida in diseased Yesso scallops examined, suggesting that in Japan, francisellosis of Yesso scallops is caused by a novel type of F. halioticida and not by the abalone type.

A new myxosporean species, Henneguya lata n. sp. (Myxozoa: Myxobolidae), from the gills of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae) in the Gulf of Tonkin, Vietnam.

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 µm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) µm, body width 6.7 ± 0.3 (6.1-7.6) µm, thickness 5.1 ± 0.2 (4.8-5.4) µm, caudal appendage length 10.0 ± 1 (8.3-11.6) µm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) µm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) µm long and 1.9 ± 0.2 (1.5-2.3) µm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

Development of a simple host-free medium for efficient prezoosporulation of Perkinsus olseni trophozoites cultured in vitro.

The parasitizing stage (trophozoite) of the protozoan parasite Perkinsus olseni progresses to the dormant stage (prezoosporangium) immediately after the death of the host through physiologically and morphologically drastic changes. This development is reproducible in Ray's fluid thioglycollate medium (RFTM). In this study, supplementation with tissue extract from a host, the Manila clam, significantly improved the efficiency of development, as determined by the numbers and sizes of developed prezoosporangia. Similar results were seen following supplementation with boiled host tissue extract, which indicates that a thermally stable component of the host is required for the parasite's development. Subsequently, we found that a commercially available lipid concentrate significantly increased prezoosporulation without host tissue, suggesting that the lipids in host tissue enhance prezoosporangia development. Moreover, we determined that yeast extract, sodium thioglycollate, and sodium chloride were the only components of RFTM required for prezoosporulation. Based on these findings, we prepared a simple, host-free medium for P. olseni prezoosporulation-Lipid concentrate Yeast extract Medium (LpcYM)-consisting of yeast extract, lipid concentrate, sodium thioglycollate, and sodium chloride. We confirmed that the prezoosporangia developed in LpcYM produce zoospores that are infectious to Manila clams and that trophozoites of other Perkinsus species (P. marinus, P. honshuensis, and P. chesapeaki) also develop to prezoosporangia in this host-free medium. As LpcYM has the simplest composition of prezoosporulation media available thus far, it enables us to conduct molecular and biochemical studies examining the drastic transformation process of this parasite.

Anisakis spp. in toothed and baleen whales from Japanese waters with notes on their potential role as biological tags.

In this study, Anisakis nematodes isolated from toothed and baleen whales from localities around Japan were molecularly (PCR-RFLP) identified. In Wakayama, common bottlenose dolphins (Tursiops truncatus) were infected with A. simplex sensu stricto (s.s.), A. typica and A. pegreffii, while A. typica was the only species found in pantropical spotted dolphin (Stenella attenuata) and striped dolphin (S. coeruleoalba). Offshore common minke whales (Balaenoptera acutorostrata) and sei whales (B. borealis) were almost exclusively infected with A. simplex s.s.. However, in common minke whales from two Hokkaido localities, mature worms mostly consisted of A. simplex s.s. in some individuals and of A. pegreffii in others, but immature worms were mainly A. simplex s.s.. Gross and histopathological examination on gastric mucosa attached by anisakids resulted in mild and superficial reactions by the two baleen whale species in contrast to severe inflammatory reaction associated with ulcer formations by common bottlenose dolphin. Host specificity and adaptability of Anisakis spp. in these baleen and toothed whales were discussed from the points of view of adult worm size, worm population and pathological reactions by hosts. Interestingly, most of the common minke whales predominantly harboring mature A. pegreffii adults belonged to the Yellow Sea - East China Sea stock (J stock), which migrates through the Sea of Japan, whereas most of those mainly parasitized by mature A. simplex s.s. adults were from the Okhotsk Sea - West Pacific stock (O stock), mostly inhabiting the Pacific side, suggesting that these sibling species may have utility as biological tags to differentiate whale stocks. These results represent the first definitive host records for A. pegreffi in the Northwestern Pacific Ocean.

A novel dimorphic microsporidian Ameson iseebi sp. nov. infecting muscle of the Japanese spiny lobster, Panulirus japonicus, in western Japan.

Japanese spiny lobsters (Panulirus japonicus) exhibiting white opaque abdominal muscle were found in Mie and Wakayama prefectures, in mid-Western Japan. Microscopically, two types of microsporidian spores, ovoid and rod-shaped, were observed infecting the muscle. Histologically, both types of spore were detected inside myofibers of the abdomen, appendages, and cardiac muscles and were often both observed in a single myofiber simultaneously. Transmission electron microscopy revealed that ovoid spores have villous projections on the surface, and that ovoid and rod-shaped spores have a polar filament with 12 coils and 6 to 8 coils respectively. Merogonic and sporogonic stages were observed around ovoid spores, but rarely around rod-shaped spores. The small subunit ribosomal DNA sequences obtained from both spore types were identical to each other, indicating that this microsporidian exhibits a clear spore dimorphism. Phylogenetic analysis based on the rDNA sequences indicates that this microsporidian is part of a clade consisting of the genera Ameson and Nadelspora, with the most closely related species being A. herrnkindi found in the Caribbean spiny lobster P. argus. Based on ultrastructural features, molecular phylogenetic data, host type and geographical differences among known species in these genera, the species found in whitened abdominal muscles of the Japanese spiny lobster is described as Ameson iseebi sp. nov.

Stable and quantitative small-scale laboratory propagation of Cryptocaryon irritans.

We established a laboratory propagation method of Cryptocaryon irritans, a parasitic ciliate of marine fishes, with black molly Poecilia sp. as host fish, using small plastic aquaria. One cycle of the propagation usually takes one week. With this method, 1500-3000 protomonts are obtained from five challenged mollies every week, from which more than 100,000-200,000 theronts are obtained. Using this method, an isolate of C. irritans has been successfully maintained more than three years. This propagation method reduces labor for maintaining and propagating the parasite and will much contribute to researches on cryptocaryoniasis.•The method is a laboratory propagation technique of Cryptocaryon irritans.•Using small plastic aquaria and black molly as a host, the parasites can be stably propagated and maintained.•An isolate of C. irritans has been successfully maintained more than three years.

Anisakis spp. in fishery products from Japanese waters: Updated insights on host prevalence and human infection risk factors.

The nematodes of the genus Anisakis are among the most relevant parasitic hazards in fishery products since they are responsible for human infection and allergy cases. In a food safety and epidemiological perspective, several marine hosts from different locations around Japan were examined to characterize the parasitism of Anisakis larvae. Chum salmon (Oncorhynchus keta) and Alaska pollock (Gadus chalcogrammus) showed the highest overall prevalence (100%), followed by blue mackerel (Scomber australasicus) (97.5%), Pacific cod (Gadus macrocephalus) (80%), chub mackerel (Scomber japonicus) (60.1%), Japanese flying squid (Todarodes pacificus) (17%) and Japanese pilchard (Sardinops sagax melanostictus) (2%). In Pacific krill (Euphausia pacifica), apart from one Hysterothylacium aduncum larva, no Anisakis specimens were detected. Anisakis simplex sensu stricto was molecularly identified (PCR-RFLP) for the first time in Japanese flying squid and Japanese pilchard distributed in the Northwestern Pacific ocean. That was the most frequent parasitic species detected followed by A. pegreffii, mostly in the western areas of Japan, hybrid genotypes between the two sibling species as well as A. typica and A. berlandi. Surprisingly, A. simplex s.s. was the most abundant species in one batch of chub mackerel from the East China Sea and A. pegreffii was the main species found in one batch from the Pacific coast of Aomori, which seems to indicate that the ranges of these two sibling species might be more variable than previously thought.

The effects of environmental and nutritional conditions on the development of Perkinsus olseni prezoosporangia.

Perkinsus olseni is a widely distributed protozoan pathogen that infects a wide range of marine mollusks. Prezoosporulation of P. olseni trophozoites is easily observed in Ray's fluid thioglycollate medium, but in nature, trophozoites within host tissue should be able to develop into prezoosporangia without any additional artificial medium after the host dies. How this process might work in field conditions remains poorly understood, however, partly because of the lack of appropriate in vitro assays. In this study, we observed that trophozoites of P. olseni successfully developed into prezoosporangia when mixed with minced tissue of the Manila clam Ruditapes philippinarum and placed in seawater. We were thus able to establish a new method to examine the development of P. olseni to prezoosporangia under artificially simulated natural environmental conditions. Using this method, we found that low temperatures (5 °C, 15 °C) significantly suppressed prezoosporangia development. In addition, we found that prezoosporangia were developed in a wide range of salinities (10-50 practical salinity unit) and that P. olseni requires some nutrition factors from host tissue for prezoosporulation to occur. Because the transmission of P. olseni among a host population highly depends on the developmental process of prezoosporangia, which leads to production of the infective zoospore stage, these results will help further our understanding of the parasite's infection dynamics in nature.

Parallel studies confirm Francisella halioticida causes mortality in Yesso scallops Patinopecten yessoensis.

Francisella halioticida is a marine bacterium originally described as the causative agent of mass mortality among giant abalone Haliotis gigantea. Recent field studies in Canada and Japan have suggested that this bacterium is also the cause of adductor muscle lesions and high mortality of Yesso scallops Patinopecten yessoensis, although a causal relationship has not been established. In the present study, the pathogenicity of F. halioticida in Yesso scallops was assessed in both Canada and Japan using bacteria isolated from diseased Yesso scallops in each respective country. Independent laboratory experiments revealed that scallops challenged with F. halioticida via bath exposure resulted in high mortality and histological lesions characterized by massive haemocyte infiltration. The presence of F. halioticida was confirmed using PCR, and F. halioticida was re-isolated from a portion of dead and surviving specimens. These results fulfill Koch's classic criteria for establishing disease causation and provide conclusive evidence that F. halioticida causes adductor muscle lesions and high mortality in Yesso scallops.

First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan.

During analyses of the invasive Mediterranean mussel Mytilus galloprovincialis for pathologies in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis was found through histological examination, Ray's Fluid Thioglycollate Medium assays, and molecular analyses. Specific PCR assays for each Perkinsus species also revealed the presence of an indigenous congeneric species, Perkinsus olseni, but P. beihaiensis was dominant in M. galloprovincialis. Sequences of the ribosomal internal transcribed spacer region I of P. beihaiensis found in Japan were genetically more similar to those found in South American countries (Panama and Brazil) than in Asian countries (China and India). Though Mediterranean mussels have become widespread in Japanese waters since their invasion in the 1930s, epidemiological surveys show that mussels collected outside Tokyo Bay are free of any Perkinsus infections. Based on these results, it was strongly suggested that P. beihaiensis invaded Tokyo Bay by transportation of bivalves originating from South America but has not yet spread to other parts of Japan. The possibility is not ruled out, however, that the parasite is indigenous in Japan but the environment in Tokyo Bay favors its transmission to Mediterranean mussels.

New insights into the entrance of Perkinsus olseni in the Manila clam, Ruditapes philippinarum.

In order to understand interactions between Perkinsus olseni and its host mollusk species Manila clam Ruditapes philippinarum, this study focused on invasion processes of the parasite, particularly the mechanisms of zoospore transformation to trophozoites in its portal entry into the host. We exposed Manila clam to P. olseni zoospores, then periodically quantified parasite intensity in various host organs and tissues. We detected large numbers of parasite cells within gills and labial palps of the host clam from the early to the final stages, moderately within mantle and digestive organs but low numbers within hemolymph, foot and adductor muscles. Our results suggest that P. olseni first invades the gills and labial palps of the host clam with limited translocation throughout the host body via the host's circulatory system until 12 days post exposure to zoospores. P. olseni zoospores exposed to extracts of gills and labial palps transformed into trophozoites more efficiently than they did when exposed to other tissues; this transformation was not observed when zoospores were exposed to heated organ extracts. Our results suggest the involvement of a host molecule in the transformation of P. olseni zoospores, leading to initial infection primarily within gills and labial palps of the host clam.